Diagnostic assay method and kit for the detection of HHV-8 infection

ABSTRACT

A method establishes for the first time a HHV-8 producing immortalized lymphoma cell line, which is free of EBV, CMV, and HIV. Large quantities of uncontaminated HHV-8 are produced by the cells, and the virus or immunogenic fragments thereof are used to obtain specific polyclonal and monoclonal antibody. Assays and kits are useful for detecting viral infection in mammalian samples.

This application is a division of U.S. patent application Ser. No.09/068,714, filed May 14, 1998, now U.S. Pat. No. 6,156,499, whichclaims the benefit of International Application No. PCT/US98/01483,filed Jan. 27, 1998, and the benefit of U.S. Provisional Application No.60/036,985, filed on Jan. 30, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a lymphoma cell line capable ofproducing large to quantities of Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8), which are substantially free from humanimmunodeficiency virus (HIV), Cytomegalovirus (CMV) and Epstein-BarrVirus (EBV). This invention also relates to a method and kit fordetecting HHV-8 infection.

2. Description of the Background

Kaposi's sarcoma (KS) is a rare neoplasm of multi focal origincharacterized by red-purple to blue-brown lesions of the skin. Cellproliferation occurs initially in the skin, eventually spreading toother body sites, in particular to the lower extremities. Lymphaticinvolvement is not unusual in KS patients, and may be present as alymphadenopathy. Kaposi's sarcoma is the most frequent neoplasticmanifestation of HIV infection, and is used as one of the criteria todecide whether an HIV-infected individual is defined as having AcquiredImmunodeficiency Syndrome (AIDS).

Four different epidemiologic forms of KS have been described: sporadicor classic KS, endemic KS, KS encountered among transplant recipientsreceiving immunosuppresive therapies, and KS prevalent among patientswith human immunodeficiency virus (HIV) infection. The “classic” form ofKS was described over a century ago in predominantly elderly men ofMediterranean and Jewish descent. Men are affected by this form of KS 10to 15 times more often than women, and those affected are typically intheir 60s or older, and have an average survival time of approximately10 years. The “endemic” form of KS has been recognized in certaingeographic regions of Central Africa. This is a neoplasm which alsoaffects men more frequently than women, is generally more aggressivethan classic KS, and involves the lymph nodes and viscera, as well. Amarked increase in the form of KS encountered in patients receivingimmunosuppressive therapy, was mostly found in hepatic and renaltransplant patients. AIDS patients have a probability of about 40% ofdeveloping cancer, especially Kaposi's sarcoma and/or non-Hodgkin'slymphoma. Kaposi's sarcoma has, additionally, been associated withlymphoid cancer in patients both with and without AIDS.

Epidemiologic studies conducted with classic KS, endemic KS, andtransplant patients suggest that both the infectious agent and theimmune status of the individual are of significance in acquiring KS. Theunidentified infectious agent, presumably the causative agent, has beenreferred to as Kaposi sarcoma-associated herpes virus (KSHV) and humanherpes virus-8 (HHV-8), interchangeably. Two novel DNA fragments werefound in 90% of KS lesions associated with AIDS. The isolation andidentification of these DNA fragments from the KS lesions of an AIDSpatient, designated KS330 and KS631, suggested the involvement of aninfectious agent. The base sequences of the two DNA fragments, and theirflanking sequences were shown to have significant homology with twoknown herpes viruses: herpes virus saimiri and Epstein-Barr Virus (EBV).The latter two viruses belong to the gammaherpesvirinae subfamily, whosemembers have the ability to replicate in lymphoblastoid cells. Of allmembers of the subclass, EBV is the best studied. EBV has been shown toinduce latent infection of peripheral blood lymphocytes in its naturalhost, and to immortalize lymphocytes in vitro, thereby causing thedevelopment of malignant lymphomas such as endemic Burkitt's lymphomas,AIDS-related non-Hodgkin's lymphomas and lymphoproliferative disorderswhich occur after transplantation. A subset of non-Hodgkin's lymphomas,referred to as body cavity-based lymphomas BCBLs, or primary effusionlymphomas (PELs), present unique clinical, morphologic,immunophenotypic, and molecular genetic characteristics. BCBLs, forinstance, grow mainly in the pleural, pericardial, and abdominalcavities, usually without an identifiable tumor mass. Thecytomorphologic features of BCBLs appear to bridge large-cellimmunoblastic and anaplastic large-cell lymphomas. Their cells are largeand possess moderate to abundant amphophilic to deeply basophiliccytoplasm, and large, round to ovoid nuclei containing one or more largenucleoli. The lymphomas have indeterminate (non-B, non-T cell)immunophenotypes, and commonly express CD45 in the absence of other B orT cell lineage-restricted antigens. At the molecular level, thelymphomas are characterized by a B-cell genotype, as determined byclonal immunoglobulin gene rearrangements, and the absence of c-myc generearrangement.

As the numbers of reported AIDS cases increased, a concomitant rise inthe incidence of KS was observed. This increase in KS has led to anincreased effort to determine the pathogenesis of the neoplasm. Theunavailability of a model system, however, has hindered these efforts.

The noticeable structural homology between EBV and KSHV has led to thehypothesis that KSHV might also be a transforming agent. In addition,KSHV/HHV-8 sequences were demonstrated in AlDS-related lymphomas, evenin the absence of KS symptoms, but could not be detected in mostnon-AIDS-related lymphoid neoplasms. Since no source of HHV-8 free ofother viruses was available, all cell lines established from neoplasmsobtained from the HIV-infected individuals failed to provide, and couldnot be used as, a model system for KSHV/HHV-8, due to co-infection byother viruses, particularly EBV.

Herpes viruses as a group establish latent infections for the entirelifetime of their host. Their DNA genomes are relatively large, 100-250kb, and may exist extra chromosomally in latently infected cells. Theactivation of an herpes virus results in viral replication, andeventually in cell lysis, with the viral copy number increasingsubstantially from the latent to the lytic infection stages. The latentstages of HSV in general, and KSHV in particular, infection produce nosymptoms. The infected individuals, however, are still capable oftransmitting the virus, and infecting others. Thus, although theKSHV/HHV-8 is present in the blood of seemingly healthy, yet infected,subjects, at present there are no acceptable methods to monitor theblood supply for HHV-8 infection.

Clearly, thus, up to the present time, the isolation andcharacterization of KSHV has been hampered because all cell sources havebeen co-infected with EBV, CMV, and/or HIV. Thus, the availability of aready, abundant, and uncontaminated source of KSHV/HHV-8 would permitthe systematic monitoring of blood bank stocks as well as of new blooddonors to avoid the spread of the virus. In addition, it would permitthe development of specific antibodies, and to conduct furtherpathogenicity studies.

SUMMARY OF THE INVENTION

The present invention, thus, relates to the isolation for the first timeof a lymphoma cell line producing large quantities of Kaposisarcoma-associated herpes virus (KSHV/HHV-8), which is substantiallyuncontaminated with human immunodeficiency virus (HIV), Cytomegalovirus(CMV) and/or Epstein-Barr Virus (EBV).

This invention also relates to a method for establishing lymphoma cells,as immortalized cell lines, and cell cultures producing copiousquantities of KSHV/HHV-8.

The present invention also relates to the HHV-8 virus, free of viralcontamination, produced by the lymphoma, and to antibody selectivelybinding HHV-8.

This invention also relates to methods and kits for screening for HHV-8infection, with the aid of purified HHV-8 virus and/or antibody bindingthe virus. These products are useful in assays and kits for thedetection of HHV-8 infection.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention arose from a desire by the inventors to develop asystem for the easy and copious production of HHV-8 for application tothe mass screening of biological samples for HHV-8 infection, forexample for monitoring blood bank stocks, and newly donated blood andtheir donors. The availability of such screening system will, in turn,permit the ready separation and discarding of contaminated blood andblood product samples and, thus, help prevent further dissemination ofHHV-8 infection, and of all diseases associated with it.

This invention, thus, provides for the first time a method ofestablishing an HHV-8 infected immortalized (established) lymphoma cellline, which is free from infection with the Epstein Barr virus (EBV),cytomegalovirus (CMV) and/or Human Immunodeficiency Virus (HIV), anHHV-8 producing cell line produced by this method, a method forproducing copious amounts of virus in the substantial absence of celllysis, and HHV-8 virus, produced by the cell line of the invention.

“KS-1” is one HHV-8 infected cell line produced in accordance with theinvention.

“HHV-8”, as used herein, means human herpes virus-8, which is synonymouswith KSHV, Kaposi sarcoma-associated herpesvirus. HHV-8 is a gamma-2herpesvirus (genus rhadinovirus), and is the first member of this genusknown to infect humans.

The phrases “body-cavity based lymphoma” or “primary effusion lymphoma”(PEL), as used herein, refer to a lymphoma that is characterized as aprimary lymphomatous effusion occurring in the pleural, pericardial,and/or abdominal cavity, usually in the absence of an identifiable tumormass. Those skilled in the art will recognize the interchangability ofother terms used to describe such lymphomas, such as, but not limitedto, primary effusion lymphomas, KSHV-associated lymphomas, and the like.

The term “pleural fluid”, as used herein, means an exudative fluidlocalized to the thoracic cavity. Pleural fluid may be collected bythoracentesis, i.e., puncturing the chest wall with a needle andcollecting the pleural fluid in a syringe by aspiration. Similar fluidmay be obtained from other serous cavities, e.g., peritoneum,pericardium, and the like.

The term “mammal”, within the context of this invention, refers to anymammal capable of being infected with HHV-8, such as mouse, rat, rabbit,non-human primates, and humans, among others. In a preferred embodimentthe mammal is a human.

The term “appropriate growth medium” means, as used herein, a mediumcomprising one or more substances that promote the growth of viablecells. Suitable growth media include, for example, RPMI, MinimumEssential Medium Eagle (MEM), and the like. The growth media aretypically supplemented with fetal bovine serum (FBS) at a concentrationof up to about 20%. Antibiotics, such as penicillin, streptomycin, andthe like, may also be added to the media to inhibit bacterialinfections. In a preferred aspect of the invention, the FBS is carefullypreselected, as is known in the art, for its ability to allow the growthof blood cells, and their progenitors.

The term “ascites-inducing amount” means, as intended herein, asufficient number of cells to produce a marked accumulation of serousfluid within a reasonable time in the ascitic cavity of a mammal.Typically, ascites fluid may be induced within about three weeks. In oneembodiment of the invention, the ascites fluid is induced within about 6weeks, preferably within about 3 weeks, and in some instances withinabout 5 days being especially preferred.

The term “immunocompromised animal”, as used herein, means an animalwith an impaired immune system such that it is incapable of fullyreacting immunologically to pathogens. Those skilled in the art willrecognize that this may be due to a genetic disorder, disease process,irradiation or drugs, such as corticosteroids or immunosuppressiveagents, given to treat a disorder that inhibits immune function.Examples of genetic disorders which inhibit immune function includeX-linked severe combined immunodeficiency, Nezelof's syndrome,Wiskott-Aldrich syndrome, leukocyte adhesion deficiency, and the like.Disease processes which inhibit immune function include disorders suchas primary or secondary bone marrow failure, lymphomas and othermalignant tumors, acquired immunodeficiency syndrome, and the like,prevent proper function of the immune system. Examples of drugs thatsuppress the immune system are methotrexate, cyclophosphamide,6-mercaptopurine, vincristine, and the like. Suitable immunocompromisedanimals for use in the practice of the present invention are the nudemouse, SCID mouse, BNX immunodeficient mouse, and the like. A mostpreferred embodiment of the immunocompromised mammal is thetriple-immunodeficient BNX mouse.

The term “immunogenic portion or fragment” means any unique portion ofthe virus capable of stimulating an immunological response, i.e.,eliciting an antibody response.

The term “antibody”, as used herein, is intended to include fragmentsthereof, as is known in the art, which also selectively bind HHV-8,including any unique portion of the virus. The antibody fragments may beobtained by fragmenting the entire antibody using conventionaltechniques, and the fragments screened in the same manner as describedabove for whole antibodies. For example, “F(ab′)₂” fragments may begenerated by treating an antibody with pepsin or papain, and “Fab”fragments may be obtained by treatment of the F(ab′)₂ fragment with areducing agent to reduce the disulfide bridges.

As used herein, the phrase “antigenic component” means either KS-1cells, HHV-8 virus, antibody binding HHV-8, and the like.

The phrase “sample” as used herein refers to any biological fluid, suchas whole blood, plasma, or other bodily fluids or tissues having eitherHHV-8 or anti-HHV-8 antibodies, preferably serum, and the like.

The present invention also provides a method for establishingimmortalized lymphoma cells, such as KS-1, in culture. The methodcomprises culturing a sample from a fluid, such as pleural, ascitic, orpericardial fluid, obtained from a body cavity of a subject afflictedwith a body cavity-based lymphoma (BCBL), or primary effusion lymphoma(PEL), in an appropriate growth medium as a primary isolate or culture,injecting an ascites-inducing amount of the primary isolate or cultureinto an immunocompromised animal, isolating cells from the ascitic fluidof the immunocompromised animal, and culturing the isolated establishedcells in an appropriate growth medium. The cell lines obtained hereindemonstrate an enhanced ability to grow in culture as compared withcells that have not been injected into immunocompromised animals, i.e.,cells from the primary isolate or culture.

Also provided herein is a method for growing large amounts of,isolating, and purifying the HHV-8 virus obtained from the cell line ofthe invention. The method generally comprises growing a lymphoma cellline prepared by the method of this invention in a growth medium,allowing the cells in culture to produce large amounts of HHV-8 virus,and separating the virus from the cells and/or the medium. Suitablepurification methods are well known to those skilled in the art. (See,for example, B. Roizman, R. J. Whitley, and C. Lopez (eds.), HumanHerpes viruses, Raven Press Limited, N.Y. (1993)). The separation may beattained by, for example, but is not limited to, centrifugation, and thepurification to affinity separation with the aid of antibody selectivelybinding to it. Other methods may also be utilized.

The purified virus obtained from the cells of this invention, e.g., theKS-1 cell line, constitutes at least about 85%, more generally at leastabout 90%, or in some instances at least about 95%, by weight of thematerial isolated from the cells. In other words, the purified virus ofthe present invention contains no greater than about 15% impurities,i.e., non-viral particles, preferably no greater than about 10%impurities, and most preferably no greater than about 5% impurities. Thepurified virus is, however, substantially free of contamination withother viral particles, and generally has an isopycnic sucrose density ofabout 1.15 g/cm³. The HHV-8 virus of this invention has been shown tocontains only human sequences and is, therefore, considered to be freeof contamination with materials from other species, including murinesequences.

The purified virus, being free of contamination from other viruses ingeneral, and particularly from HIV, CMV, and EBV, is particularlysuitable for the preparation of antibodies selectively binding HHV-8, oran immunogenic fragment thereof. For example, anti-serum or monoclonalantibodies which selectively bind the virus or one of its fragments, maybe obtained by methods known in the art using the an attenuated form ofthe entire virus, an immunogenic fragment or a capsid protein thereof.Briefly, a mammal, such as, a mouse, hamster, or rabbit, may beimmunized with an immunogenic form of HHV-8, such as an inactivatedHHV-8, a capsid protein, and the like. The technology for conferringimmunogenicity on a protein or peptide are known in the art. Forexample, the protein or peptide may be administered in the presence ofan adjuvant to enhance and amplify its immunogenicity. Furthermore, onceobtained, the polyclonal or monoclonal antibodies of the presentinvention may be used to isolate, and further purify naturally occurringHHV-8. Those of skill in the art will readily identify suitableadjuvants for use herein, given that such materials are well known inthe art. Following effective immunization, HHV-8 anti-serum may beobtained and, if desired, polyclonal anti-HHV-8 antibodies isolatedtherefrom. Hybridoma cell lines secreting monoclonal antibodies (mAbs)selectively binding the HHV-8 virus may be obtained by the Kohler andMilstein technology, and other techniques, such as the EBV-hybridomatechnique to produce human monoclonal antibodies, and the human B-cellhybridoma technique. Briefly, antibody producing cells, i.e.,lymphocytes, are harvested from an animal immunized as described above,and fused by, for example, standard somatic cell fusion procedures withimmortalizing cells, such as myeloma cells, preferably of non-producingmyeloma cells, to yield the immortalized hybridoma cells. Suchtechniques are well known in the art. (Kohler & Milstein, Nature (1975)256: 495-497; Cole et al., Monoclonal Antibodies & Cancer Therapy (1985)Alan R. Less, Inc., pp. 77-96; Kozbar, et al., Immunology Today (1983)4:72). The thus obtained hybridoma cells may then be immunochemicallyscreened for the production of antibodies selectively binding theisolated HHV-8, or a portion or fragment thereof, and the monoclonalantibodies isolated by affinity separation.

The antibodies of the invention selectively binding HHV-8 or portions orfragments thereof may also be utilized as a tool in diagnostic assays,which are suitable for application to all tissues, such as a biopsy, orto a fluid sample, such as, serum, obtained from a subject. Highlyspecific serological tests are of extreme value in, for example,screening populations for HHV-8 infected subjects, monitoring the bloodbank supplies, and the like. The utilization of the present diagnosticmethod and kit, will lead to ensuring, for the first time, a safe, i.e.,HHV-8-free, blood supply which, in turn, will reduce the likelihood oftransmission of the virus.

The methods for detecting HHV-8 infection in a sample, in accordancewith this invention, are varied, and include those relying on thedetection of the virus or a portion thereof, and those relying on thedetection of antibody elicited by viral infection. The overall methodsrely on known technologies, such as immunohistochemistry, in situhybridization, fluorescent in situ hybridization, radioimmunoassay, andthe like, and may include labels such as radiolabels, enzymes,fluorescent, and phosphorescent labels, and the like (DeLuca,“Immunofluorescence Analysis”, in Antibody as a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp 189-231 (1982), the relevantportions of which are incorporated herein by reference). One method fordetecting the presence of antibody-bound complexes employs an“ELISA”-type assay, that relies on the detection and quantification ofeither antibody or antigen (depending on the ELISA format type) presentin a sample. ELISA assays are a well-known techniques that may bereadily carried out by those having ordinary skill in the art (Chapter22, 4th Edition of Basic and Clinical Immunology, D. P. Sites et al.,Lange Medical Publication, CA (1982), the relevant portions of whichbeing incorporated herein by reference). ELISA assays employed in thepractice of the present invention include the use of the antibody and/orvirus of this invention, or of fragments of either one or both, or ofthe cell line of the invention, preferably bound to a solid matrix orsupport which, as is well known in the art, are water insoluble andinclude crosslinked dextran (Pharmacia Fine Chemicals; Piscataway,N.J.), agarose, polystyrene beads (typically about 1 micron to about 5millimeters in diameter (Abbott Laboratories, Chicago, Ill.), cellulose,polyvinyl chloride, polystyrene, crosslinked polyacrylamide,nitrocellulose- and nylon-based webs such as sheets, strips or paddles,or tubes, plates, or wells of a microtiter plate, such as those madefrom polystyrene or polyvinylchloride, and the like. The antibodies ofthe invention may be labeled, to facilitate the identification of HHV-8or antibody selectively binding the virus in a sample, with a variety ofdetectable compounds. The detectable label may be a fluorescent labelingagent that chemically binds to the antibody without producingdenaturation to form a fluorochrome (dye) useful as an immunofluorescenttracer. Suitable fluorescent labeling agents include fluorochromes, suchas fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamino-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride, and the like. Radioactive atoms are also usefuldetectable labels for antibodies, including those which produce gammaray emission, such as ¹²⁵I and ¹³¹I. The antibody label may also be anenzyme, such as horseradish peroxidase (HRP), glucose oxidase, alkalinephosphatase, and the like. In this case, e.g., HRP or glucose oxidase,additional reagents are typically required to indicate that anantibody-antigen complex has formed, which include hydrogen peroxide,and an oxidation dye precursor such as o-phenylenediaminedihydrochloride, diaminobenzidine, and the like. An additional reagentuseful with glucose oxidase is2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid), and one usefulwith alkaline phosphatase is para-nitrophenyl phosphate. Depending onthe nature of the label or catalytic signal producing system used, theproduced signal may be detected by irradiating the complexed test samplewith light of the correct wavelength and observing the level offluorescence, by contacting the complexed sample with a substrate whichmay be catalytically converted by the label to produce a dye,fluorescence, phosphorescence, or chemiluminescence, in which theformation of dye may be observed visually or in a spectrophotometer, byemploying a radiation counter such as a gamma counter to detect gammaemitting labels such as ¹²⁵I, and the like.

The methods described above, and others as well, may be practiced withthe kits of this invention for detection of HHV-8 infection in a subjectusing anti-HHV-8 antibodies described herein. The kits which, inaccordance to the invention, may be applied in the detection of HHV-8infection may contain, for example, the antibodies described above,monoclonal or polyclonal, or their fragments, which selectively bindHHV-8 or a portion therof, which may be conjugated to a fluorochrome, aphosphochrome, enzyme, or radiolabel, an appropriate substrate forenzyme-linked antibodies, e. g., hydrogen peroxide for a peroxidase,blocking solutions, e.g., normal goat or rabbit serum, 3% bovine serumalbumin in physiological saline, and the like, and buffers, e.g.,Tris-HCl, phosphate buffered saline, EDTA, and the like, among others,as well as combinations of any two or more thereof.

The HHV-8 obtained by the method of the invention may also be utilizedin a kit for diagnosing HHV-8 infection in a subject. The substantiallypure HHV-8 of this invention may be attached to a solid support, andcontacted with the blood or blood product of a subject, to detect anyantibodies selectively binding to HHV-8 present in the subject's bloodor blood product, by binding to the immobilized HHV-8, and the boundcomplex detected with, e.g., a labeled secondary antibody such asanti-human IgG, fragments thereof, or protein A or G. Another kitprovided herein is useful for the detection of anti-HHV-8 antibodies ina sample, and may contain the virus or an antigenic component thereof,immobilized onto a slide or microtiter plate, anti-human antibodies orfragments thereof, or protein A or G, buffers, and a detectable (labeledor to be labeled) secondary antibody. The secondary antibodies,polyclonal and preferably monoclonal, useful in the practice of thepresent invention may be obtained by techniques well known in the art.The polyclonal antibodies may be obtained, for example, by the methodsin Ghose et al., Methods of Enzymology, 93, 326-327 (1983). with IgG orFe fragments of IgG being used as the immunogen to stimulate theproduction of IgG-reactive polyclonal antibodies in the antisera ofnon-human animals, such as rabbits, goats, sheep, rodents, and the like.Depending on the secondary antibody or label used, the kits mayoptionally contain a signal generating substance to provide or enhancethe detection of the anti-HHV-8 antibodies. Suitable substrates forenzymatic signal generation systems, include simple chromogens andfluorogens such as para-nitrophenyl phosphate, hydrogen peroxide, andthe like. In addition to the above, other components such as ancillaryreagents may be included, for example, stabilizers, buffers, e. g.,Tris-HCl, phosphate buffered saline, EDTA, and the like, fixatives, andthe like. The reagents may be provided as dry powders, usuallylyophilized, including excipients, which on dissolution will provide areagent solution having the appropriate concentrations for performingthe methods of the present invention.

The invention will now be described in greater detail by reference tothe following non-limiting examples. The relevant portions of allreferences, patents, and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLES Example 1

Establishment of KS-1 Cells in Culture

Cells from the pleural fluid from a subject manifesting primaryKSHV-positive effusion-based lymphoma were obtained by thoracentesis andgrown in RPMI plus 20% bovine serum at 37° C. in 5% carbon dioxide andair. The cells began to proliferate actively within 3 days of initial invitro culture. Cells grew nonadherently, occasionally in small clumps,and had a cellular doubling rate of about 48 to 72 hours.

Cells from the above described in vitro cultures were separated from thegrowth medium by centrifugation, washed 3 times, and resuspended insaline. The resuspended cells (4×10⁶ cells total) were injected into theperitoneal cavity of triple-immunodeficient BNX mice (supplied byHarland Sprague-Dawley in Cambridge, Mass.), who developed prominentascites within 3 weeks of injection.

Ascitic fluid was collected from the mice, centrifuged, washed, andresuspended in RPMI containing 20% fetal bovine serum (Gibco, BRL,Gaithersburg, Md.). The cells grow non-adherently with a doubling timeof approximately 48 hours.

Example 2

Immunophenotypic Characterization of KS-1 Cells

Immunophenotypic studies were performed using antibodies and/or in situhybridization probes to immunoglobulin kappa and lambda chains, CD3,CD20, CD45, CD79a (DAKO Corporation, Carpinteria, Calif.), CD5(Becton-Dickinson Immunocytometry Systems, Mountain View, Calif.), CD56(N-CAM; Bender Med-Systems, Vienna, Austria), Herpes simplex virus types1 and 2, EBV latent membrane protein (LMP-1), and EBV EBER1 (DAKO).

These immunophenotypic studies showed KS-1 cells to be positive for CD45and CD20, but negative for surface IgGs, for T-cell markers (CD3 andCD5), for CD56 (N-CAM), and for DC79a. The B-cell lineage was confirmedby the demonstration of clonal rearrangements of the Ig gene in Southernblots. In addition, the cells express recognizable human karyotipicabnormalities, and the requisite cell surface antigens. The cellsobtained from the murine ascitic fluid stained positively forcytoplasmic light chains, and were negative for EBV as evidenced byimmunohistochemistry for EBV latent membrane protein (LMP-1), and insitu hybridization for Epstein-Barr encoded RNA (EBER). The cells werealso negative for CMV, as determined by in situ hybridization, and forherpes simplex virus 1 and 2, as determined by immunohistochemistry.

Example 3

Purification of HHV-8

KS-1 cells are collected by centrifugation for 10 minutes at 1,500 rpm,resuspended in 1:20 vol of culture medium, and snap-frozen to obtain acell virus extract. A supernatent containing the virus is obtained bycentrifugation of a 1,400×g cleared conditioned culture medium for 2hours at 23,000×g at 4° C., and the pellets are then resuspended in 1:50vols of culture medium and snap-frozen. Both preparations, cell andsupernatent virus, are then thawed, sonicated to rupture the cellmembrane, cleared by centrifugation at 10,000×g at 4° C., loaded on aphosphate-buffered saline-25% sucrose cushion, and pelleted by overnightcentrifugation at 70,000×g at 4° C. The viral pellets are resuspended inPBS and prepared for further use.

Example 4

Immobilization of KS-1 Cells on Glass Slides

The cells present in the cell suspension are counted using a microscopeand hemacytometer, and resuspended in a sufficient volume of buffer toachieve 5×105 cells per ml. A sufficient volume is added to each slideso that 50,000-100,000 cells are applied to each slide.

The cells were centrifuged in a Cytospin 3™ (Shandon, Inc. Pittsburgh,Pa.) at 500 rpm for 5 minutes to apply 0.01 ml of the resuspended cellsto each slide. Cytospins are air dried for 10 minutes. The cells arethen fixed to the slide by incubation for 2 minutes in sufficient volumeof ice cold methanol/acetone (50/50) to cover the sample, and the slideis then allowed to air dry. The slides may be stored at −20° C.

Example 5

Immunofluorescence Assay

Four slides prepared in accordance with Example 4 are used for eachserum sample. Each slide is thawed, transfered to 1×PBS, blocked byincubation with 3% BSA in PBS for 30 minutes, and washed 3 times in PBS,10 minutes per wash. The slides are then dried with a Cytospin filter.

The serum to be tested is serially diluted 1:10, 1:40, and 1:160 withPBS containing 10% rabbit serum, 2% BSA and 1% glycine, and eachdilution is applied to only one slide. Only a secondary antibody isapplied to the fourth slide, which is then used as a control.

50 μl of each of the dilutions of the human serum are added to theappropriate slide, and only the secondary antibody to the control slide.The samples are incubated for 90 minutes at room temperature insufficient humidity to prevent volume loss. The sera are rinsed off withtwo 10 min. washes in PBS containing 0.4% Tween® 20, and one 10 min.wash in PBS, and the slides are dried with a Cytospin filter. 50 82 lrabbit anti-human IgG FITC antibodies (secondary antibodies) (DAKO) areadded at a 1:40 dilution in PBS to each slide, and the samples areincubated for 45 minutes at room temperature, in sufficient humidity tominimize volume loss. The slides are then rinsed three times with PBScontaining 0.4% Tween® 20, and one drop of Aquamount is applied onto theslides, and then a coverslip. The slides are stored at −20° C. in alight tight box until ready to read.

The fluorescence is read under a fluorescence microscope at 400×magnification at an excitation wavelength is 495 nm, and an emissionwavelength is 525 nm.

Example 6

Enzyme-linked Immunosorbent Assay

50 μl human sera are diluted as desired with PBS containing 10% rabbitserum, 2% BSA, and 1% glycine, and added to each slide prepared inaccordance with Example 4 by immobilizing KS-1 cells. 50 μl PBS withalbumin are added to blank slides. All samples are allowed to stand atroom temperature for one hour, in sufficient humidity to minimize volumeloss, the serum is aspirated, and the samples are washed three timeswith 50 μl of PBS containing 0.02% sodium azide (NaN3) and 0.05% Tween®.

50 μl of an alkaline phosphatase-coupled goat anti-human IgC antibody inPBS containing 0.25% bovine serum albumin is then added to each slide,the optimal dilution being pre-determined using a standard titrationassay. Goat F(ab′)₂ anti-human IgG(Fc)-alkaline phosphatase may beobtained from Jackson Immuno-Research Laboratories in West Grove, Pa.The samples are incubated for 60 to 90 minutes at room temperature insufficient humidity to minimize volume loss, and the samples washedthree times with a sufficient volume of PBS containing 0.02% sodiumazide (NaN3) and 0.05% Tween® to cover the cells. All samples are thenwashed three more times with TRIS-NaCl solution containing 0.05M Tris,0.15M NaCl, and 0.02% sodium azide, pH 7.5, and 0.75 g disodiump-nitrophenol phosphate (United States Biochemicals catalogue #19587 orAMRESCO catalogue #P0364) is combined with a Tris buffer containing 75mM Tris-HCl, 1.5 mM MgCl2, 0.02% sodium azide, pH 8.6 to form asubstrate containing solution, 50 μl of which are added to each slide.The samples are incubated at room temperature for about 30 to 90 minutesin sufficient humidity to minimize volume loss, and the slide read at405 nm.

The KS-1 cell line was deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A. 20852,on Dec. 18, 1996, under the terms of the Budapest Treaty on theInternational Recognition of Deposits of Microorganisms for Purposes ofPatent Procedure and the Regulations promulgated under this Treaty. TheKS-1 cell line has been given ATCC Accession No. CRL-12247. Samples ofthe deposited material are, and will be made, available to industrialproperty offices and other persons legally entitled to receive themunder the terms of the Treaty and Regulations and, otherwise, incompliance with the patent laws and regulations of the United States ofAmerica and all other nations or international organizations in whichthis application, or an application claiming priority of thisapplication, is filed, or in which any patent is granted on any suchapplication. In particular, all restrictions upon the availability ofthe deposited material will be irrevocably removed upon issuance of aU.S. patent based on this, or any application claiming priority to, orincorporating this application by reference thereto.

Those having ordinary skill in the art will recognize, or be able toascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. Suchequivalents are considered to be within the scope of this invention andare covered by the following claims.

What is claimed is:
 1. An assay method for detecting antibodyselectively binding HHV-8, comprising: contacting with HHV-8 viralparticles a serum sample originating from an individual suspected ofhaving an HHV-8 infection, said HHV-8 viral particles having beenproduced by and isolated from immortalized lymphoma cells, wherein thecells are from a cell line deposited with ATCC as Accession No.CRL-12247, said lymphoma cells originating from cells isolated andcultured from ascitic fluid of an immunocompromised animal, said animalhaving previously received into its peritoneal cavity an inoculation ofan ascites-inducing number of cells from a primary cell culture, saidprimary cell culture having been derived from the pleural fluid of acavity-based lymphoma of a HHV-8⁺ human, said human at the time ofproducing the pleural fluid being HIV⁻, CMV⁻, and EBV⁻; allowing theformation of complexes of the antibody and the HHV-8 viral particles;contacting any antibody-bound HHV-8 complexes thus formed withanti-human antibody-binding immnunoglobulin or anti-humanantibody-binding fragments thereof, said fragments being selected fromthe group consisting of Fab and F(ab′)₂, and allowing the formation ofcomplexes of the immunoglobulin or the fragments thereof with theantibody-bound HHV-8 complexes; and detecting the presence of anycomplexes formed.
 2. An anti-HHV-8 antibody detecting kit, comprising:HHV-8 viral particles, said HHV-8 viral particles having been producedby and isolated from immortalized lymphoma cells, wherein the cells arefrom a cell line deposited with ATCC as Accession No. CRL-12247, saidlymphoma cells originating from cells isolated and cultured from asciticfluid of an immunocompromised animal, said animal having previouslyreceived into its peritoneal cavity an inoculation of anascites-inducing number of cells from a primary cell culture, saidprimary cell culture having been derived from the pleural fluid of acavity-based lymphoma of a HHV-8⁻ human, said human at the time ofproducing the pleural fluid being HIV⁻, CMV⁻, and EBV⁻; and labeledanti-human antibody-binding immunoglobulin or labeled anti-humanantibody-binding fragments thereof selected from the group consisting ofFab and F(ab′)₂.
 3. The anti-HHV-8 antibody detecting kit in accordancewith claim 2, further comprising: a solid matrix for supporting saidHHV-8 viral particles.